Les auteurs sont soutenus par le Conseil de recherche suédois, la Fondation suédoise pour la recherche stratégique, la société du cancer suédois, la société suédoise de l&#39;enfant le cancer, Wibergs Åke fondation et Jeanssons fondation.

1. Introduire la sonde à cellules <ol><li> Co-transfection de cellules avec une sonde FRET base et une résistance plasmidique conférant. Choisissez une méthode de transfection qui est efficace dans votre cellule-type d&#39;intérêt. Pour les cellules U2OS, les méthodes classiques de transfection de phosphate de calcium donnent des résultats adéquats 15. </li><li> Sélectionnez les cellules dans l&#39;antibiotique approprié pendant au moins sept jours. Ceci enrichit la quantité de cellules exprimant la sonde et limite la quantité de cellules avec des niveaux d&#39;expression toxiques, ou les niveaux d&#39;expression qui affectent sévèrement le cycle cellulaire. </li><li> (Facultatif) Sélectionnez des clones stables. </li><li> En utilisant des cellules fixes ou en direct, vérifier que les deux CFP et YFP sont présents dans toutes les cellules à peu près le même ratio. </li><li> Dans le cas de certaines cellules ne contiennent que des CFP ou YFP, la recombinaison a probablement eu lieu. La recombinaison peut se produire pendant la croissance bactérienne ou après transfection de la sonde et peut dans ce dernier cas dépendra de la qualité de plasmide. Répétez plasmide préparation et de linéariser le plasmide avant la transfection, si le problème persiste. </li></ol> 2. Ratiométrique surveillance FRET <ol><li> Cellules semences sur des plats à fond de verre, ou des lamelles pour le montage dans une chambre. Assurez-vous que les plats / lamelles sont pas 1.5 (170 um d&#39;épaisseur). </li><li> Mont cellules dans un milieu de croissance sans rouge de phénol sur un microscope à épifluorescence motorisée avec contrôle de température réglée sur 37 ° C. Assurer la régulation du pH des médias, soit par le CO 2 ou en utilisant le CO 2-médias indépendants tels que Liebowitz-15. </li><li> Focus sur les cellules en utilisant la lumière transmise. Ne cherchez pas sur les cellules en utilisant la lumière fluorescente. </li><li> Commencez par acquérir une image de 12 ou 16 bits en utilisant une excitation YFP (YFPex) et YFP émission (YFPem) filtres et d&#39;un miroir dichroïque adaptée. Utilisez binning maximal disponible qui permet encore la résolution requise subcellulaire. L&#39;utilisation d&#39;un binning haute pour diminuer le temps d&#39;exposition ou l&#39;intensité est cruciale pour éviter la phototoxicité. </li><li> MeAsure ou estimer l&#39;intensité du bruit de fond et approximative en mesurant ou en estimant la moyenne approximative, les valeurs minimales et maximales des pixels dans une zone dépourvue de cellules. Changer le temps d&#39;exposition et les filtres de densité neutre de sorte que la moyenne du signal dans la cellule est d&#39;environ de l&#39;intensité de fond et de cinq à dix fois la différence entre max et l&#39;intensité de base min. </li><li> Vérifiez que les images ne sont pas près d&#39;être saturé. L&#39;intensité maximale de l&#39;image doit être inférieure à la moitié de la plage dynamique (par exemple max 2000 en une image 12 bits) pour permettre des différences d&#39;intensité lorsque les cellules entrent mitose. </li><li> Répétez l&#39;étape 2.4 à 2.6 en utilisant une excitation PCP et filtres d&#39;émission YFP et un miroir dichroïque adaptée. </li><li> Sélectionner plusieurs régions avec des cellules transfectées, en excluant les régions utilisées pour le point 2.4 à 2.7, et de s&#39;assurer que l&#39;intensité maximale est inférieure à la moitié de la plage dynamique dans toutes les cellules. </li><li> Démarrer une expérience time-lapse d&#39;acquérir deux images toutes les 45 min à l&#39;aide YFPex - YFPem et CFPex - YFPem jeux de filtres. </li></ol> 3. Vérification et optimisation des conditions <ol><li> Ouvrez les images acquises et de surveiller le temps du cycle cellulaire de la mitose à la mitose des cellules qui expriment la sonde transfectées. </li><li> Comparez le temps cellulaire mesurée cycle à données publiées pour le type de cellules utilisées. Alternativement, le film non transfectées cellules par l&#39;acquisition d&#39;une image de la lumière transmise uniquement (DIC par exemple ou en contraste de phase) toutes les heures pour obtenir des temps de référence du cycle cellulaire. </li><li> Dans le cas du temps de la mitose à la mitose correspond à la référence horaires du cycle cellulaire, passez à l&#39;étape 4. Alternativement, ré-acquérir des images en utilisant davantage l&#39;exposition, plus de temps-points ou de multiples niveaux et z répétez l&#39;étape 3. </li><li> Dans le cas du temps de la mitose à la mitose ne correspond pas à référencer les horaires du cycle cellulaire, le film de cellules transfectées par l&#39;acquisition d&#39;une image de la lumière transmise uniquement (par exemple, DIC ou à contraste de phase) toutes les heures suivies d&#39;une image à la finde l&#39;expérience pour identifier les cellules transfectées. </li><li> Si les cellules transfectées montrent encore les temps de cycle plus long cellulaire, répétez l&#39;étape 1, mais moins transfecter sonde ou de sélectionner des clones stables qui contiennent des quantités très faibles de la sonde. </li><li> Si les cellules transfectées montrent les temps normaux du cycle cellulaire en l&#39;absence de lumière fluorescente, répétez l&#39;étape 2 mais inférieur à l&#39;exposition en modifiant binning, les filtres de densité neutre, le temps d&#39;exposition, et le temps entre les images. </li></ol> 4. L&#39;analyse de FRET <ol><li> L&#39;analyse peut être effectuée par la plupart des logiciels dédiés à la microscopie. Nous décrivons ici comment effectuer l&#39;analyse en utilisant le logiciel gratuit ImageJ ( <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> ) en utilisant le plug-in Ratio Plus ( <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus . html</a> ). </li><li> Ouvrez les images dans ImageJ. Dans le cas de vos images sont dans le format d&#39;un STAC multicolork (par exemple DeltaVision), séparer les canaux individuels d&#39;abord en les convertissant en une HyperStack: Image-HyperStack-pile pour HyperStack et ensuite les partager aux piles utilisant un seul canal: Image-HyperStack-réduire la dimensionnalité </li><li> Multipliez le YFPex - pile YFPem avec 3 à l&#39;aide: Processus de-Math-Multiply Cette étape est facultative, car elle est uniquement destinée à accroître la clarté visuelle des images en faisant en sorte que les ratios ne sont pas dans la gamme entre 0 et 1. </li><li> Dans le YFPex - pile YFPem, dessiner une région d&#39;intérêt (ROI) dans une zone qui est dépourvu de cellules, mais qui est proche d&#39;une cellule pour être mesurée. Différentes cellules dans la même image peut nécessiter différentes régions, à la fois comme arrière-plan et de l&#39;intensité du signal est plus fort en général au centre d&#39;une image. </li><li> Ajouter le ROI d&#39;un retour sur investissement responsable: Analyser-tools-ROI gestionnaire. </li><li> Mesurer l&#39;intensité moyenne du ROI dans les deux YFPex - YFPem et le CFPex - pile YFPem: Analyser-Mesure </em&gt;. </li><li> Répéter à dosages répétés et vérifier que l&#39;intensité de base proche de la cellule d&#39;intérêt sont similaires tout au long du film. </li><li> Set mesures pour y inclure l&#39;intensité minimale: Analyser Set mesures-Min et Max grise de valeur. </li><li> Dessinez une zone d&#39;intérêt couvrant la plupart d&#39;une cellule et mesurer l&#39;intensité minimale dans les deux YFPex - YFPem et le CFPex - pile YFPem. Prenez la différence entre l&#39;intensité mesurée minimale et l&#39;intensité de fond de 4,6 et le diviser par deux. Cela donne une estimation de départ pour une valeur d&#39;écrêtage. </li><li> Ratio plus ouvert. Sélectionnez le YFPex - YFPem et le CFPex - pile YFPem. Pour FRET rapport, sélectionnez CFPex - YFPem que la pile 1, pour le FRET inversé ratio de visualiser les sondes FRET où diminue l&#39;efficacité lors de la phosphorylation, sélectionnez YFPex - YFPem que la pile 1. </li><li> Insérer les intensités de fond mesurée et les valeurs d&#39;écrêtage. </li><li> Réglez la mise à l&#39;échelle de la pile ratio qui en résulte et d&#39;appliquer un u look adaptép table (LUT) pour visualiser changements de rapport. </li></ol> 5. Les résultats représentatifs PLK1 l&#39;activité est d&#39;abord visible dans le noyau en phase G2 et les sommets lors de la mitose. La figure 3 montre une expérience en utilisant les paramètres de phototoxicité minimale telle que décrite dans la section 2. S&#39;il vous plaît noter que ceci est un résultat représentatif initial et que les conditions d&#39;exposition ou de temps entre les images peuvent être modifiées pour augmenter rapport signal-bruit ou de la résolution temporelle. La figure montre en 3D que la majorité des cellules exprimant la sonde prolifèrent avec un temps de cycle cellulaire entre 20 et 25 heures, indiquant que les conditions d&#39;imagerie et de niveaux d&#39;expression de la sonde n&#39;affectent pas les horaires du cycle cellulaire. Bien qu&#39;il y ait beaucoup de bruit, la tendance des PLK1 activité croissante dans G2 et un pic dans la mitose 14 est clairement visible (figure 3A). Traitement des données brutes, ici signifie filtrage présenté comme un kymographe (figure 3B), ou la quantification de la moyenne de la PR inversé<html> tio (figure 3C) peut améliorer la clarté. <img alt="Figure 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> Figure 1. Principe d&#39;une sonde FRET basée à surveiller les activités kinases et de phosphatases. Deux fluorophores, généralement CFP (bleu) et YFP (en vert), sont reliés par un domaine phospho-contraignante (orange) et une séquence phosphorylable (jaune). Phosphorylation (rouge) médie liant au domaine phospho-contraignante, ce qui induit un changement conformationnel dans la sonde. Les résultats changement conformationnel dans une différence dans la distance ou l&#39;orientation entre les deux fluorophores, qui affecte le FRET efficacité entre CFP et YFP. FRET peut être visualisé par PCP excitante et la surveillance des émissions de YFP (lignes pointillées). <img alt="Figure 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> Figure 2. Aperçu schématique de la procédure expérimentale. p_upload/3410/3410fig3.jpg &quot;/&gt; Figure 3. PLK1 l&#39;activité est d&#39;abord visible dans le noyau en phase G2 et les sommets lors de la mitose. Cellules exprimant un U2OS FRET basée sur la surveillance de sonde PLK1 activités ont été filmées pour 9,14 60 h en utilisant un système d&#39;imagerie DeltaVision Spectris équipé d&#39;un objectif 20x NA air 0,7 et une lampe au mercure. Conditions d&#39;imagerie ont été choisis pour provoquer la phototoxicité minime, tel que décrit dans l&#39;article 2, en utilisant binning 4x et filtres à densité neutre qui bloquent 99% de la lumière entrante. Une représentation, en fausses couleurs de FRET-ratio inversé, à la suite d&#39;une cellule à 4 divisions. B, kymographe de la cellule représentée en A après avoir appliqué un filtre moyenneur. C, la quantification de l&#39;inversion de FRET rapport de la cellule représentée en A. D, l&#39;entrée en mitose cumulé de 50 FRET sonde cellules exprimant, y compris les divisions des cellules filles.

<ol>
	<li>Sun, Y., Wallrabe, H., Seo, S. A., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+microscopy+in+2010:+the+legacy+of+Theodor+Forster+on+the+100th+anniversary+of+his+birth.">FRET microscopy in 2010: the legacy of Theodor Forster on the 100th anniversary of his birth.</a> Chemphyschem. 12, 462-474 (2011).</li><li>Allen, M. D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochem. Biophys. Res. Commun. 348, 716-721 (2006).</li><li>Kunkel, M. T., Ni, Q., Tsien, R. Y., Zhang, J., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+dynamics+of+protein+kinase+B/Akt+signaling+revealed+by+a+genetically+encoded+fluorescent+reporter.">Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter.</a> J. Biol. Chem. 280, (2005).</li><li>Violin, J. D., Zhang, J., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+reporter+reveals+oscillatory+phosphorylation+by+protein+kinase.">A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase.</a> C. J. Cell. Biol. 161, 899-909 (2003).</li><li>Kunkel, M. T., Toker, A., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+regulation+of+protein+kinase+D+revealed+by+a+genetically+encoded+kinase+activity+reporter.">Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter.</a> Journal of Biological Chemistry. 282, 6733-6742 (2007).</li><li>Harvey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+sensor+of+ERK+activity.">A genetically encoded fluorescent sensor of ERK activity.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 19264-19269 (2008).</li><li>Fosbrink, M., Aye-Han, N. N., Cheong, R., Levchenko, A., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+JNK+activity+dynamics+with+a+genetically+encoded+fluorescent+biosensor.">Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5459-5464 (2010).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+activation+of+CyclinB1-Cdk1+coordinates+entry+to+mitosis.">Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis.</a> Dev. Cell. 18, 533-543 (2010).</li><li>Fuller, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midzone+activation+of+aurora+B+in+anaphase+produces+an+intracellular+phosphorylation+gradient.">Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient.</a> Nature. 453, 1132-1136 (2008).</li><li>Ni, Q., Titov, D. V., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+kinase+dynamics+in+living+cells+with+FRET+reporters.">Analyzing protein kinase dynamics in living cells with FRET reporters.</a> Methods. 40, 279-286 .</li><li>Morgan, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The cell cycle : principles of control. , (2007).</li><li>Lindqvist, A., Rodriguez-Bravo, V., Medema, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decision+to+enter+mitosis:+feedback+and+redundancy+in+the+mitotic+entry+network.">The decision to enter mitosis: feedback and redundancy in the mitotic entry network.</a> J. Cell. Biol. 185, 193-202 (2009).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cyclin+B1-Cdk1+synchronizes+events+in+the+nucleus+and+the+cytoplasm+at+mitosis.">Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.</a> J. Cell. Biol. 189, 247-259 (2010).</li><li>Macurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polo-like+kinase-1+is+activated+by+aurora+A+to+promote+checkpoint+recovery.">Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.</a> Nature. 455, 119-123 (2008).</li><li>van der Eb, A. J., Graham, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+transforming+activity+of+tumor+virus+DNA.">Assay of transforming activity of tumor virus DNA.</a> Methods. Enzymol. 65, 826-839 (1980).</li><li>Lamprecht, M. R., Sabatini, D. M., Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+free,+versatile+software+for+automated+biological+image+analysis.">CellProfiler: free, versatile software for automated biological image analysis.</a> Biotechniques. 42, 71-75 (2007).</li><li>Roszik, J., Lisboa, D., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intensity-based+ratiometric+FRET+in+image+cytometry--approaches+and+a+software+solution.">Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.</a> Cytometry A. 75, 761-767 (2009).</li></ol>

Nous n&#39;avons rien à révéler.

Surveillance de FRET au long du cycle cellulaire nécessite des considérations qui sont moins cruciales lors de l&#39;évaluation à court terme des réponses à des stimuli externes. Tout d&#39;abord, la progression du cycle cellulaire est facilement perturbée par la signalisation du stress, exigeant que la phototoxicité est maintenu à un minimum. Deuxièmement, tous les journalistes peuvent potentiellement affecter les processus cellulaires en titrant sur les kinases, les phosphatases ou des domaines d&#39;interac...

<table border="1"><tbody><tr><td> Réactif </td><td> Numéro de catalogue </td><td> Société </td></tr><tr><td> Leibovitz L-15, pas de rouge de phénol </td><td> 21083-027 </td><td> GIBCO, par Life Technologies </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO, par Life Technologies </td></tr><tr><td> Sérum fœtal bovin (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0,05% de trypsine EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Pénicilline-streptomycine </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO, par Life Technologies </td></tr><tr><td> Puromycine </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster résonance de transfert d&#39;énergie (FRET) basé sur une reporters permettre l&#39;évaluation de la kinase endogène et activités phosphatase dans des cellules vivantes. Ces sondes sont généralement constitués de variantes du CFP et YFP, est intervenu par une séquence phosphorylable et un domaine phospho-contraignant. Après phosphorylation, la conformation sonde change, ce qui entraîne un changement de la distance ou l&#39;orientation entre le CFP et YFP, conduisant à un changement dans l&#39;efficacité de FRET (Fig 1). Plusieurs sondes ont été publiés durant la dernière décennie, la surveillance de l&#39;équilibre d&#39;activité des kinases et phosphatases multiples, y compris les journalistes de la PKA 2, PKB 3, 4 PKC, PKD 5, 6 ERK, JNK 7, CDK 18, Aurora B 9 et 9 PLK1 . Compte tenu de la conception modulaire, les sondes supplémentaires sont susceptibles d&#39;émerger dans le proche avenir 10. La progression dans le cycle cellulaire est affectée par le stress signaling voies 11. Notamment, le cycle cellulaire est régulée différemment pendant imperturbable croissance par rapport à quand les cellules se remettent du stress 12. Imagerie time-lapse de cellules dans le cycle cellulaire nécessite donc des précautions particulières. Cela devient un problème particulier lors de l&#39;utilisation d&#39;imagerie ratiométrique, puisque deux images avec un signal élevé par rapport au bruit sont nécessaires pour interpréter correctement les résultats. Ratiométrique FRET imagerie des modifications du cycle cellulaire dépendante des activités kinase et phosphatase a essentiellement été limité à des sous-sections du cycle cellulaire 8,9,13,14. Ici, nous discutons d&#39;une méthode pour surveiller FRET basée sur des sondes à l&#39;aide d&#39;imagerie ratiométrique au long du cycle cellulaire humain. La méthode repose sur l&#39;équipement qui est disponible pour de nombreux chercheurs en sciences de la vie et ne nécessite pas de connaissances d&#39;experts de la microscopie ou de traitement d&#39;image.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET basée sur les journalistes sont de plus en plus utilisées pour suivre les activités kinases et de phosphatases dans les cellules vivantes. Nous décrivons ici une méthode sur la façon d&#39;utiliser FRET basé journalistes afin d&#39;évaluer le cycle cellulaire dépendante des changements dans la phosphorylation cible.

Activités de surveillance kinase et la phosphatase dans le cycle cellulaire par Ratiométrique FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K vues.  Karolinska Institutet. FRET basée sur les journalistes sont de plus en plus utilisées pour suivre les activités kinases et de phosphatases dans les cellules vivantes. Nous décrivons ici une méthode sur la façon d'utiliser FRET basé journalistes afin d'évaluer le cycle cellulaire dépendante des changements dans la phosphorylation cible.

Vidéo: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Dessin de sang de rats à travers la veine saphène et par ponction cardiaque

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Recherche

Biologie

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

À long terme des essais de toxicité létale avec le Crustacé Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET microscopie pour Surveillance en temps réel des événements de signalisation dans les cellules vivantes en utilisant Biocapteurs unimoléculaires

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Étude de l&#39;ADN Looping par une seule molécule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Synchronisation de<em&gt; Caulobacter crescentus</em&gt; Des enquêtes sur le cycle cellulaire bactérienne

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Approches expérimentales pour l&#39;étude mitochondrial Localisation et fonction d&#39;une cellule du cycle nucléaire Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Conformations GPCR G Protein-sélectifs mesurée à l&#39;aide de FRET capteurs dans un Fluorometer Assay de cellules vivantes de suspension

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinant la synchronisation cellulaire mitotique et microscopie confocale haute résolution pour étudier le rôle des protéines multifonctionnelles Cycle cellulaire au cours de la mitose

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...